Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

• the present invention relates to methods of predicting those individuals likely to develop persistent infection after exposure to the hepatitis virus, particularly the hepatitis B virus.
• cytokine IL10 (also known as cytokine synthesis inhibitory factor) is produced by TH2 cells, a subset of T cells which favour antibody production (Roitt, Bostoff & Male-fifth Edition, Mosby).
• IL10 inhibits the production of the IFN-gamma, by inhibiting the development of interferon secreting lymphocytes (THl lymphocytes). It also inhibits the production of the cytokines IL-1, IL-6 and TNF-alpha by macrophages, and favours antibody type immune responses during infection.
• THl lymphocytes are thought to be essential for the control of viral replication and the elimination of hepatocytes infected with the hepatitis B virus (Penna et al. , Hepatology, 25(4): 1022-7 (1997)).
• patients infected with the virus may be treated with either interferon alpha or lyphoblastoid interferon.
• the response rate for this therapy is limited, e.g only around 40% in the case of chronic HBV.
• IL10 1082G* There is a point mutation at position 1082 (with respect to the transcriptional start site), (IL10 1082G*) which appears to be of functional significance: An adenine to guanine substitution is associated with increased levels of IL10 secretion (Turner et al., Eur. J. Immunogenet : , 24(1): 1-8 (1997)).
• IL10 1082 A* allele low IL10 secretion level
• IL10 1082 G* is associated with the clearance of HBN. This affects the prognosis or treatment of an individual patient subject to HBV infection.
• gamma interferon could also be expected to influence the outcome not only of hepatitis B infection, but also hepatitis C, hepatitis G, human papilloma virus, human immunodefiency virus and other persistent virus infections.
• the present invention provides, a method for predicting the outcome of a virus infection in a subject, comprising the step of determining whether the subject carries one or more alleles associated with altered clearance of said virus.
• predicting the outcome of a virus infection means predicting the susceptability of a subject to infection by a virus (following exposure) and/or predictng the susceptability of a subject to suffer disease/damage as a result of infection.
• allelic variation in the present context means that the allelic variation is associated with an alteration in the natural or normal clearance rate of the virus. This may occur as a result of an altered secretion of the cytokine, for instance.
• the present invention provides a method for predicting the outcome of a virus infection in a subject, comprising the step of determining whether the subject carries one or more alleles associated with altered secretion of a cytokine.
• the virus infection is a hepatitis virus infection, particularly hepatitis B.
• the method comprises determining whether the subject carries the IL 10 A* allele, or the ILIOG* allele.
• the preferred method of carrying out the determination is to analyse a sample of the subject's DNA.
• a sample can conveniently be obtained from a biological sample, e.g. blood or a tissue sample.
• the subject is preferably a human.
• the DNA obtained from the biological sample will be amplified using techniques well known to those skilled in the art, e.g. PCR techniques (Sambrook et al, Molecular Cloning, third edition - Cold Spring Harbor Labs Press,).
• PCR techniques Standardbrook et al, Molecular Cloning, third edition - Cold Spring Harbor Labs Press,
• IL10 gene region and more particularly the IL10 promoter region can be amplified.
• Such techniques will involve the use of at least one pair of suitable primers.
• Suitable primers can be chosen on the basis of the DNA sequence coding for the cytokine in question.
• suitable primers include the following:
• These primers are designed to amplify a 656bp sequence of the DNA that includes the ILIO 1082 point mutation.
• the presence of the point mutations will be detected using a sequence specific oligonucleotide hybridisation technique, as described herein.
• a sequence specific oligonucleotide hybridisation technique will involve the use of suitable probes which will be chosen on the basis of the DNA sequence coding for the cytokine in question.
• suitable probes include the following:
• IL10 gene region can mean the whole of the IL10 gene, or, alternatively, a part thereof. Clearly, however, if only a part is amplified it should include that portion of the gene associated with a particular point mutation, polymorphism etc. For instance in the case of the IL10 1082A*/IL1082G* allele, the portion of the gene which is amplified must include the promoter and may also include the coding region.
• the present invention provides nucleic acid sequences comprising at least one of the sequences as set out in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, or a fragment thereof comprising at least nine nucleotides.
• the nucleic acid sequence is one which hybridises to a flanking region of an allele associated with virus infection.
• the allele is associated with infection by hepatitis, in particular hepatits B.
• the present invention provides a kit for use in a method for predicting the outcome of a virus infection in a subject which comprises one or more reagents for use in determining the presence or absence of one or more alleles associated with altered clearance of the virus.
• a reagent includes one or more primers.
• the present invention provides a kit for use in a method for predicting the outcome of a virus infection in a subject which comprises at least one pair of primers suitable for PCR amplification of at least a portion of the gene coding for a cytokine, and/or at least one pair of probes suitable for oligonucleotide hybridisation to the cytokine DNA sequence.
• hybridisation means that one oligonucleotide sequence will specifically anneal to a complementary oligonucleotide sequence and will remain annealed under stringent conditions, for example, at 35 to 65° C in a salt solution of about 0.9M .
• PCR primers and conditions The primers are designed to amplify a 656bp sequence of DNA that includes all three of the point mutations. This fragment of the promoter region of human IL-10 gene, spanning -1179 to -523, was amplified by PCR with the use of
• the reaction mix contains:
• a sequence specific oligonucleotide hybridisation technique is used to identify the genotype.
• the procedure can now be repeated, using the same filter, with the second digoxigenin labelled probe, and films for the two alleles compared and the genotype recorded.
• Blocking reagent stock (Boehringer Mannheim) 10ml 10% laurylsarcosine Water to make 1000ml.
• TMAC hybridisation solution 600ml 5M TMAC 50ml IM Tris pH 8 10ml 10% SDS
• Buffer 2 50ml lOx buffer 1
• Stripping buffer 2 100ml 20x SSC 10ml 10% SDS Water to 1000ml
• ILIO 1082 A* allele (low secretion level) was associated with persistent infection in two totally independent populations. We therefore conclude that IL10 1082G* is associated with clearance of HBV.

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• 1998
  • 1998-08-07 GB GBGB9817266.1A patent/GB9817266D0/en not_active Ceased
• 1999
  • 1999-08-09 CA CA002339526A patent/CA2339526A1/en not_active Abandoned
  • 1999-08-09 WO PCT/GB1999/002603 patent/WO2000008215A1/en not_active Application Discontinuation
  • 1999-08-09 AU AU54295/99A patent/AU5429599A/en not_active Abandoned
  • 1999-08-09 EP EP99940297A patent/EP1102866A1/de not_active Withdrawn
• 2001
  • 2001-02-07 US US09/777,924 patent/US20020106745A1/en not_active Abandoned

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